Turnover of glucose and acetate coupled to reduction of ... - Biodeep

FEMS Microbiology Ecology 31 (2000) 73^86

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Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice ¢eld soil Amnat Chidthaisong, Ralf Conrad * Max-Planck-Institut fu«r terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany Received 30 March 1999 ; received in revised form 6 October 1999; accepted 9 October 1999

Abstract Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-14 C]glucose and [2-14 C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N2 O). Addition of nitrate resulted in complete inhibition of CH4 production from [U-14 C]glucose and [2-14 C]acetate. However, addition of ferrihydrite or sulfate decreased the production of 14 CH4 from [U-14 C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of 14 CO2 from [U-14 C]glucose, but all increased the production of 14 CO2 from [2-14 C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced 14 CH4 production from [2-14 C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO3 2 and N2 O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH4 towards CO2 production. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Methane; Turnover; Threshold; Competition ; Radiotracer;

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1. Introduction Complete mineralization of organic carbon such as carbohydrates to gaseous products in anaerobic environments requires the co-operation of several microbial populations [1]. Generally, hydrolytic microorganisms are responsible for conversion of polymers to monomers and oligomers in the ¢rst step. The products of this depolymerization, such as mono- and oligosaccharides, are then used for cell and energy production by these bacteria. Under anaerobic conditions, however, various fatty acids, H2 and alcohols are produced. The fatty acids larger than acetate are then further metabolized by proton-reducing bacteria to H2 and acetate [2,3]. The ¢nal step in organic carbon mineralization involves the conversion of acetate and H2 either to CH4 in methanogenic environments or to CO2 when

* Corresponding author. Tel. : +49 (6421) 178 801 ; Fax: +49 (6421) 178 809; E-mail : [email protected]

C; Paddy soil; Chloroform

inorganic electron acceptors such as nitrate, ferric iron or sulfate are available. In rice ¢eld soil, decomposition of organic matter serves as an important electron source for those bacteria which use nitrate, ferric iron, sulfate or CO2 as electron acceptors in the anaerobic respiration process. According to the thermodynamic theory, electron acceptors with a higher redox potential are reduced ¢rst [4,5]. As a result, it is predicted that nitrate is to be reduced ¢rst, followed by ferric iron, sulfate and CO2 . Thus, principally, there will be no CH4 production until reduction of nitrate, ferric iron and sulfate is complete. However, the situation in nature is more complicated. For example, sequential reduction can only operate if corresponding bacterial physiotypes are active. Simultaneous reduction of ferric iron, sulfate and production of methane may also be possible, provided that each bacterial population is active and substrates serving as electron donors are present in excess [6^8]. The availability of electron donors is the most impor-

0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 9 ) 0 0 0 8 3 - 5

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A. Chidthaisong, R. Conrad / FEMS Microbiology Ecology 31 (2000) 73^86

tant factor controlling which reduction process is dominant. In rice ¢eld soil, competition for available H2 has been relatively well documented [8^10]. It seems that nitrate reducers are able to use H2 e¤ciently and thus outcompete not only methanogens for H2 but also ferric iron reducers and sulfate reducers [8,10]. Likewise, ferric iron reducers and sulfate reducers can outcompete methanogens for H2 [8]. As a result, H2 -dependent methanogenesis was inhibited during reduction of nitrate, ferric iron and sulfate [8^10]. The reason for the superior competition of nitrate reducers versus ferric iron reducers, sulfate reducers and methanogens is seen in the thermodynamics of the redox reactions resulting in a successively increasing threshold for H2 [11^14]. Theoretically, competition for acetate should be similar to H2 . However, our knowledge is mainly based on investigation of pure cultures and environments other than rice ¢eld soil [15^18]. A comparative study of the competition for acetate by nitrate reducers, ferric iron reducers, sulfate reducers and methanogens is lacking altogether. This knowledge is important for rice ¢eld soil, where all these redox processes simultaneously take place in the rhizosphere [19]. However, the few observations that exist are not always conclusive. Addition of nitrate to anoxic paddy soil results in complete inhibition of methanogenesis, but does not result in a decrease of the acetate pore water concentration, indicating that inhibition is caused by toxic e¡ects of the denitri¢cation intermediates nitrite, nitric oxide (NO) and N2 O rather than by competition for acetate [10,20,21]. Addition of ferrihydrite or sulfate to anoxic rice ¢eld soil also results in more or less complete inhibition of methanogenesis [8,9], but the experimental evidence for the mechanism of inhibition is contradictory. Achtnich et al. [9] observed in Italian paddy soil that ferric iron reducers did not have a lower threshold for acetate than methanogens. On the other hand, Sigren et al. [22] observed in Texas rice ¢elds that acetate concentrations decreased during drainage concomitantly with an increase of ferric iron and a decrease of CH4 emission, suggesting that ferric iron reducers decreased the acetate concentration below the threshold of methanogens. Achtnich et al. [9] reported that addition of sulfate resulted in the complete inhibition of CH4 production, but not in any decrease of the acetate concentration. Since acetate-utilizing sulfate reducers were only present as spores and in low numbers, the authors suggested that acetate consumption was due to interspecies H2 transfer between acetate-utilizing methanogens and H2 -utilizing sulfate reducers [9,23]. On other occasions, however, acetate-utilizing Desulfotomaculum species were found to germinate in Italian rice ¢eld soil after 13 weeks of incubation and are then present in numbers as high as H2 -utilizing sulfate reducers [24]. In addition to Desulfotomaculum species, Italian rice ¢eld soil also seems to contain acetate-utilizing sulfate reducers related to the genus Desulforhabdus [25]. Even less is known about microbial metabolism and

competition of more complex organic substrates such as glucose. Recently, we have demonstrated that glucose is quantitatively the most important monosaccharide in rice ¢eld soil [26]. Glucose uptake is rapid and glycolysis is the main degradation pathway [27]. In addition, glucose serves as an important precursor for acetate production [26]. Since species of nitrate reducers (e.g. [28]), ferric iron reducers (e.g. [29]) and sulfate reducers (e.g. [30]) exist which are able to utilize hexose, it is possible that these species compete with fermenting bacteria for glucose and that it is this competition which results in the inhibition of CH4 production rather than the competition with methanogens for acetate and H2 . The objective of the present study was to test this hypothesis by investigating the turnover of radioactive glucose and to study the competition for acetate during the reduction of nitrate, ferric iron and sulfate and the production of CH4 . We used anoxic rice ¢eld soil as methanogenic model system. The soil slurries were prepared from dry soil which was sampled from drained and fallow Italian rice ¢elds. The preparation of anoxic soil slurries from crushed soil lumps mimics the £ooding of the plowed and harrowed ¢elds at the beginning of the rice-growing season. 2. Materials and methods 2.1. Soil sample and slurry incubation Rice ¢eld soil was taken in 1993 from the experimental ¢eld of the Italian Rice Research Institute in Vercelli. Detailed site descriptions and soil characteristics were already given in a previous study [31]. The air-dried soil was mechanically crushed and sieved ( 6 0.5-mm mesh size). Soil slurries were prepared by adding 28 ml of distilled and sterilized water to 28 g dry soil in a sterile 120-ml serum bottle, giving a ¢nal volume of the soil slurry of 32.7 ml. The bottles were then closed with sterile black rubber stoppers and crimped with aluminum caps. The gas head space was exchanged with N2 . To obtain methanogenic steady state conditions, the soil slurry was pre-incubated at 30³C for 36 days. During this period, the concentrations of nitrate, ferric iron, sulfate, H2 and CH4 were monitored. Depletion of nitrate, ferric iron and sulfate, establishment of the stable concentrations of H2 and acetate and a linear increase in CH4 were considered as the criteria for steady state methanogenesis (Fig. 1). This soil preparation was done three times in sequence, i.e. for measuring the sequential reduction processes (Fig. 1), for measuring glucose turnover and for measuring acetate turnover. The experimental bottles were incubated at 30³C without shaking to avoid damage of the methanogenic community [32]. At given time intervals, gas samples (1 ml) were taken from the head space after vigorously shaking

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the bottles by hand and then analyzed for H2 , CO2 and CH4 . Each experiment was set up with numerous replicate bottles which were sequentially terminated at given time intervals by addition of 3 ml 7 N H2 SO4 and then used for analysis of dissolved compounds. Liquid samples (1 ml) were taken and centrifuged at 14 000Ug for 5 min. The supernatant was membrane-¢ltered (0.2 Wm, polytetra-

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£uoroethylene; Sartorius, Go«ttingen, Germany) and stored frozen (320³C) until analysis. To study glucose and acetate turnover coupled with di¡erent reduction processes, the soil slurry was supplemented with the following compounds as electron acceptors: sodium nitrate (10 mM); ferrihydrite, Fe(OH)3 (50 mM) and sodium sulfate (6.25 mM). The control

Fig. 1. Temporal change of the concentrations of (A) nitrate and sulfate, (B) ferric and ferrous iron, (C) acetate and propionate and of (D) the partial pressure of H2 and the total amounts of CH4 and CO2 in freshly prepared anoxic slurries of Italian rice ¢eld soil.

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Fig. 2. Degradation of [U-14 C]glucose in 36-day old methanogenic rice ¢eld soil after previous addition of nitrate, ferrihydrite or sulfate, shown by (A,B) the decrease of [U-14 C]glucose, (C,D) the intermediate accumulation of radioactive acetate, (E,F) the accumulation of 14 CO2 and (G) the accumulation of 14 CH4 , (A,C,E,G) in the absence and (B,D,F) in the presence of chloroform. Mean þ S.D., n = 2; note the exponential x-axis.

bottles were prepared without supplement. All treatments were duplicates. Reduction of these electron acceptors was then followed by measuring their residual concentrations. Ferrihydrite was prepared by precipitation of FeCl3 in alkali as described by Schwertmann and Cornell [33]. It was used as aqueous suspension. The amount of each oxidant (electron acceptor) added was equal in terms of electron equivalents required for complete reduction (i.e. 50 mili-electron equivalents). In some experiments, chloroform (CHCl3 ) was added to a ¢nal concentration of 100 WM to study its e¡ect on glucose, acetate and CH4 metabolism. CHCl3 was added 1^8 h before addition of radioactive glucose or acetate.

2.2. Radioactive experiments with [U-14 C]glucose and [2-14 C]acetate Addition of the carrier-free [U-14 C]glucose or [2-14 C]acetate (purity s 99%, speci¢c activity = 11.1 and 2.11 GBq mmol31 , respectively; American Radiolabeled Chemical) was made at 3 days after the reduction of each electron acceptor (nitrate, ferrihydrite or sulfate) had started. The amount of radioactivity added to each bottle was ca. 4.5^10 Mdpm (60 Mdpm = 1 MBq). Recovery of [U-14 C]glucose from autoclaved soil slurry was relatively high and constant throughout the experiment at 97.7 þ 2.3% ( þ S.E.M., n = 4), indicating that most of the

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Fig. 2. (continued)

added [U-14 C]glucose was biologically available. The recovery of [2-14 C]acetate was also constant throughout the experiment, but was less (46.1 þ 1.6%, þ S.E.M., n = 4) than for glucose, indicating that part of the [2-14 C]acetate added was adsorbed to soil particles and/ or exchanged with acetate pools that could not be recovered by the extraction method. The same phenomenon has been reported earlier [34^36]. When used below, the term radioactivity of glucose or acetate refers to the values corrected by the radioactive recovery. 2.3. Analytical techniques Concentrations of CH4 , CO2 , N2 O and H2 were measured in the gaseous headspace of the incubated bottles by

standard gas chromatography as previously described [37,38] using a £ame ionization detector (CH4 , CO2 ), electron capture detector (N2 O) and HgO to Hg vapor conversion detector (H2 ). NO was analyzed in a chemoluminescence NOx analyzer as described [39]. Termination of the soil incubations by slurry acidi¢cation allowed us to determine the total CO2 pool [40] and resulted in the release of 14 CO2 from dissolved radioactive bicarbonate and carbonate to the gas phase. When used below, 14 CO2 represents total 14 CO2 (CO2 +bicarbonate+carbonate). Radioactivity of gaseous products was measured in a gas chromatograph equipped with a gas proportional counter as described previously [41]. Nitrate, nitrite and sulfate were determined in liquid samples by high pressure liquid chromatography (HPLC) with conductivity and UV detectors

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[42]. Organic acids and their radioactivities were determined by HPLC with a radioactivity detector [27]. The pool size of glucose was determined separately in a parallel soil incubation using PAD-HPLC [26]. Ferric and ferrous iron were analyzed after extraction of 0.5 ml soil slurry with 4.5 ml 1 N HCl using a recently developed ion chromatographic system [43]. 2.4. Calculations The uptake rate constant (k) of glucose and acetate was estimated from a semi-exponential plot of the decrease of radioactive glucose or acetate with incubation time [44]. The data points (n = 3^10) used for the calculation of glucose and acetate uptake rate constants were usually obtained within 20 and 60 min after the addition of [U-14 C]glucose and [2-14 C]acetate, respectively. The correlation coe¤cients of the linear regression were usually s 0.9 (P 6 0.05). The turnover rates of glucose and acetate were obtained by multiplying the uptake rate constant with the pore water concentration of the respective substrate. A pore water concentration of 1 WM is equivalent to 1 nmol g31 dry soil. The rates are given per g dry soil. The respiratory index (RI) was used to compare the carbon £ow towards CH4 and CO2 : RI = (14 CO2 )/ (14 CO2 +14 CH4 ). 3. Results 3.1. Processes during pre-incubation Nitrate and nitrite were only detected in freshly prepared slurries of air-dried soil (approximately 4 mM and 2 WM, respectively). Nitrate and nitrite disappeared rapidly within 1 day of anaerobic soil incubation (Fig. 1A). Thus, the rate of nitrate consumption was at least 160 nmol g31 h31 . Reduction of ferric iron also started immediately and stopped at around day 5 when the accumulation of ferrous iron reached a plateau (Fig. 1B). The rate of ferric iron consumption during days 0^4 was about 500 nmol g31 h31 . The pore water concentration of sulfate ¢rst increased during the phase of ferric iron reduction and decreased when reduction of ferric iron had ¢nished. Sulfate reduction continued until day 7 at a rate of about 40 nmol g31 h31 and ¢nished after about 10^12 days. The initial increase of sulfate in the pore water was probably due to the desorption of sulfate from ferric iron minerals ([45^47] ; Ja«ckel and Schnell, in preparation). Acetate accumulated up to about 2 mM at day 10 and its concentration stabilized around 100^200 WM during methanogenesis (Fig. 1C). Propionate increased with a lag phase of about 1 week, then accumulated up to a maximum of 60 WM at day 15 and subsequently decreased to below the detection limit of 5 WM in pore water (Fig. 1C). Other organic acids such as caproate and

lactate were also occasionally detected but at a much zzlower concentration than acetate and they were no longer detectable after the onset of methanogenesis (data not shown). H2 accumulated immediately at the beginning of anoxic incubation and its partial pressure reached s 20 Pa within 1 day (Fig. 1D). Subsequently, H2 partial pressures rapidly decreased to a relatively constant level of approximately 2.5 Pa after day 15. Linear increase in CH4 concentration was observed after all the other sequential reduction processes had ¢nished at around day 10 (Fig. 1D). The production rate of CH4 from day 10 to 36 was 36 nmol g31 h31 . On the other hand, CO2 production started right after the beginning of anoxic incubation with a rate of 150 nmol g31 h31 (from day 0 to 5, Fig. 1D). Trace amounts of CO ( 6 30 Pa partial pressure equivalent to a total amount of 6 40 nmol per g dry soil) were also detected during the ¢rst week of incubation, but the CO concentration became very low and could not be quanti¢ed thereafter (data not shown). In general, the patterns of CH4 production and change in concentrations of dissolved compounds were similar to those observed before [10]. 3.2. Activation of nitrate, ferric iron and sulfate reduction in methanogenic soil After incubation under anaerobic conditions for 36 days, nitrate, ferrihydrite and sulfate were added during the methanogenic phase. Soon after their addition, reduction of these electron acceptors was observed (data not shown). Nitrite, NO and N2 O accumulated to maximum concentrations of 85, 30 and 960 WM, respectively. The rates of the induced nitrate, ferric iron and sulfate reduction in the methanogenic soil slurry were 64, 230 and 32 nmol g31 h31 , respectively. 3.3. Turnover of [U-14 C]glucose In a preliminary experiment, when [U-14 C]glucose was added together with each electron acceptor (nitrate, ferrihydrite, sulfate), it was consumed within 20 min so that it was depleted before reduction of the added electron acceptors became obvious. Hence, the products and turnover parameters of glucose consumption were not a¡ected by addition of the electron acceptors. We therefore repeated the experiment, but the labelled glucose was added 3 days later, when reduction of the respective electron acceptor was already active. When [U-14 C]glucose was added to the soil slurry, most of it was rapidly consumed within 20 min regardless of whether nitrate, ferric iron, sulfate or chloroform was added (Fig. 2A and Table 1). In these treatments, the turnover time of glucose was usually less than 10 min. The glucose turnover rates were between 22 and 26 nmol g31 h31 , when assuming that the glucose pool size (3.3 WM) was the same as in a parallel experiment [25] and was not

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Table 1 Uptake rate constants and recovery of [U-14 C]glucose in methanogenic rice ¢eld soil and during reduction of nitrate, ferrihydrite and sulfate Treatment

Uptake rate constant (h31 )

Maximum recovery of 14 C as acetate (%)

Maximum recovery of 14 C as CO2 (%)

Maximum recovery of 14 C as CH4 (%)

Final recovery of 14 C as acetate+ CO2 +CH4 (%)

Final RI value

Control Control+CHCl3 Nitrate Nitrate+CHCl3 Ferrihydrite Ferrihydrite+CHCl3 Sulfate Sulfate+CHCl3

7.95 þ 0.06a 5.06 þ 0.21b 3.04 þ 0.03c 3.80 þ 0.18c 6.96 þ 0.24d 7.50 þ 0.18a;d 8.97 þ 0.15e 7.03 þ 0.25d

45 66 4 7 44 50 39 66

45 30 47 52 60 66 45 23

20 0 0 0 6 0 7 0

65 96 47 52 66 66 52 89

0.70 1.00 1.00 1.00 0.90 1.00 0.86 1.00

Mean þ S.D., n = 2. Di¡erent letters indicate signi¢cant di¡erences (P = 0.01) tested by ANOVA. RI = 14 CO2 /(14 CO2 +14 CH4 ).

signi¢cantly altered by the addition of ferric iron or sulfate. On the other hand, glucose uptake was partially inhibited (60% compared to the control) when nitrate was added, resulting in a glucose turnover rate constant of 3 h31 or a glucose turnover time of about 20 min (Table 1). Acetate was the ¢rst product of glucose degradation in all treatments (Fig. 2C). Other radioactive fatty acids were not observed. Accumulation of acetate usually continued for 20^40 min before being consumed. On the other hand, formation of 14 CO2 (Fig. 2E) and 14 CH4 (Fig. 2G) took a relatively longer time to reach the accumulation maxima, typically in the range of hours. The relative later production of radioactive CO2 compared to acetate was probably due to the slowly equilibrating radioactive pools [48,49]. The maximum recoveries of radioactivity as labelled acetate are summarized in Table 1. The radioactive acetate accumulated in the unamended control bottles was not di¡erent from that in the ferrihydrite bottles. Sulfate addition caused a slight decrease in the accumulation of labelled acetate. With nitrate addition, however, accumula-

tion of labelled acetate was strongly reduced and was detectable only shortly after [U-14 C]glucose addition. In all treatments with exogenous electron acceptors, the labelled acetate was eventually consumed and disappeared within 2 h after glucose addition. Addition of chloroform resulted in the accumulation of radioactive acetate in the control and the sulfate bottles (Fig. 2D). Consumption of radioactive acetate in the control did not resume throughout the experimental period, but in the bottles supplemented with sulfate, a slow consumption of acetate was observed when the incubation was prolonged. These results indicate that acetoclastic methanogenesis was completely, and sulfate-dependent acetate consumption was largely, inhibited by chloroform. With nitrate or ferrihydrite, chloroform had no e¡ect on radioactive acetate accumulation. The presence of nitrate, ferrihydrite or sulfate during the experiment with radioactive glucose resulted in a slight decrease of the acetate pool size which was generally higher than about 100 WM (Fig. 3A). Chloroform addition

Fig. 3. Temporal change of the acetate pools size during the degradation of [U-14 C]glucose shown in Fig. 2, (A) in the absence and (B) in the presence of chloroform.Mean þ S.D., n = 2. Note the exponential x-axis.

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Fig. 4. Degradation of [2-14 C]acetate in 36-day old methanogenic rice ¢eld soil after previous addition of nitrate, ferrihydrite or sulfate and of chloroform, shown by (A,B) the decrease of [2-14 C]acetate, (C,D) the accumulation of 14 CO2 and (E,F) the accumulation of 14 CH4 . Mean þ S.D., n = 2. Note the exponential x-axis.

resulted in an increase of the acetate concentrations in the control and sulfate-treated bottles, but not in the bottles treated with ferrihydrite or nitrate (Fig. 3B). In the control, radioactive CH4 was ¢rst detected about 40 min after glucose addition. However, it took about 2 h to detect 14 CH4 production in the treatments with ferrihydrite and sulfate (Fig. 2G). Clearly, CH4 production was inhibited by addition of ferric iron and sulfate. However, the inhibition of methanogenesis by these electron acceptors was not complete. Small amounts of 14 CH4 were still detected (methanogenesis was inhibited by 70 and 65% in the presence of ferric iron and sulfate, respectively; Table 1). By contrast, addition of nitrate completely inhibited

production of 14 CH4 from [U-14 C]glucose. Although addition of the di¡erent electron acceptors a¡ected the production of 14 CH4 , there was no clear e¡ect on the production of 14 CO2 , except perhaps in the presence of sulfate which caused a slightly longer lag phase of CO2 accumulation (Fig. 2E). Recovery of radioactivity as acetate, CH4 plus CO2 in these treatments generally ranged from 47 to 66% (Table 1). The missing radioactivity was probably assimilated into microbial biomass with di¡erent e¤ciencies among the di¡erent microbial physiotypes. In the control and the sulfate bottles, addition of chloroform greatly reduced the production of 14 CO2 (by 30^50% compared to the

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81

Fig. 4. (continued)

controls, Fig. 2F and Table 1). However, part of the 14 CO2 missing in the treatments with chloroform was compensated by accumulation of radioactive acetate (Fig. 2D). Thus, it appeared that the di¡erence in 14 CO2 production from [U-14 C]glucose was partly due to the inhibition of acetate consumption by chloroform. At the end of the experiment, the recoveries of labelled glucose in the products (acetate+CO2 +CH4 ) ranged from 52^96% with the lower recoveries in those treatments that were not inhibited by chloroform. 3.4. Turnover of [2-14 C]acetate Consumption of [2-14 C]acetate added to the di¡erently treated soil slurries is shown in Fig. 4. There was a lag of about 20 min for acetate uptake in the control (Fig. 4A), while in the other treatments, acetate consumption started immediately. In the nitrate-supplemented bottles, [2-14 C]acetate was consumed within 10 min (Fig. 4A) and the acetate turnover rate constant was 33 times higher than

in the control (Table 2). Compared to the control, addition of ferric iron and sulfate also resulted in an accelerated acetate consumption (Fig. 4B). In general, addition of the di¡erent electron acceptors resulted in acetate turnover times of less than 1 h, whereas in the methanogenic control, the turnover time of acetate was about 5 h (Table 2). Addition of chloroform signi¢cantly inhibited acetate consumption in the control and the sulfate bottles. Chloroform had no e¡ect on acetate consumption by nitrate reducers and iron reducers. The pool sizes of acetate in the control bottles were only slightly lower (100^150 WM) in this experiment (with [2-14 C]acetate) than in the experiment with [U-14 C]glucose (150^250 WM). However, in contrast to the experiments with [U-14 C]glucose, the acetate concentration in the experiments with [2-14 C]acetate decreased below the detection limit of the HPLC detector (5 WM) in the presence of nitrate and sulfate and in the presence of ferrihydrite acetate, it was at about 25 WM. Similarly as in the experiment with [U-14 C]glucose, acetate concentrations increased in

Table 2 Uptake rate constants and recovery of [2-14 C]acetate in methanogenic rice ¢eld soil and during reduction of nitrate, ferrihydrite and sulfate Treatment

Uptake rate constant (h31 )

Maximum recovery as 14 CO2 (%)

Maximum recovery as 14 CH4 (%)

Final recovery of 14 C as 14 CO2 +14 CH4 (%)

Final RI value

Control Control+CHCl3 Nitrate Nitrate+CHCl3 Ferrihydrite Ferrihydrite+CHCl3 Sulfate Sulfate+CHCl3

0.210 þ 0.03a 0.003 þ 0.001a 6.84 þ 0.33b 7.08 þ 0.12b 1.56 þ 0.12b 1.89 þ 0.03c 1.26 þ 0.24c 0.006 þ 0a

12 3 47 58 77 74 67 1

78 0 0 0 13 0 6 0

90 79 47 58 90 74 73 63

0.13 1.00 1.00 1.00 0.86 1.00 0.91 1.00

Mean þ S.D., n = 2. Di¡erent letters indicate signi¢cant di¡erences (P = 0.01) tested by ANOVA. The values of the control and the control+CHCl3 were signi¢cantly di¡erent by a paired t-test. RI = 14 CO2 /(14 CO2 +14 CH4 ).

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the presence of CHCl3 in the control and sulfate-amended bottles (data not shown). Radioactive CO2 was the sole product of [2-14 C]acetate utilization in the nitrate bottles (Fig. 4C). In the presence of nitrate, about 47^58% of the added radioactivity was recovered as 14 CO2 , regardless of the chloroform addition (Table 2). More 14 CO2 was observed with ferric iron and sulfate (Fig. 4D). At the end of experiment, the total recovery of [2-14 C]acetate as gaseous products ranged from 47 to 90% (Table 2). Chloroform strongly inhibited the production of 14 CO2 from [2-14 C]acetate in the presence of sulfate (Fig. 4D), and also slightly in the control (Fig. 4C), but had no e¡ect in the other incubations. In the control, most of the added [2-14 C]acetate was converted to 14 CH4 as shown by the low RI value of 0.13 (Table 2). However, addition of nitrate completely inhibited 14 CH4 production (Fig. 4E). Similarly to the 14 CH4 production from [U-14 C]glucose, small amounts of 14 CH4 were also produced from [2-14 C]acetate in the presence of sulfate (Fig. 4F). However, the inhibition of methanogenesis by sulfate was more pronounced with acetate (92%) than with glucose (65%) as substrate. Chloroform generally inhibited the production of 14 CH4 from [2-14 C]acetate (Fig. 4E,F) 4. Discussion Previous studies have shown that nitrate reducers, ferric iron reducers and sulfate reducers successfully compete with methanogens for H2 , thus inhibiting CH4 production when nitrate, ferric iron or sulfate is added to methanogenic rice ¢eld soil [8^10]. Here, we have shown that nitrate reducers, but not ferric iron reducers or sulfate reducers, were also able to utilize glucose more e¤ciently than fermenting bacteria, thus resulting in a decrease of acetate formation and in abolition of CH4 production (Fig. 2 and Table 1). Furthermore, we have shown that nitrate reducers, ferric iron reducers and sulfate reducers all successfully competed for [2-14 C]acetate that was more e¤ciently converted to 14 CO2 in the presence of nitrate, ferrihydrite or sulfate than to 14 CH4 in the unamended control (Fig. 4 and Table 2). However, we also noted that nitrate reducers may have caused toxic e¡ects by production of nitrite, NO and N2 O and that the competition for acetate did not generally result in a decrease of the acetate concentration, thus requiring complex interactions between the di¡erent microbial populations to explain our results. Previous studies have demonstrated that addition of nitrate or the denitri¢cation intermediates nitrite, NO and N2 O inhibits methanogenesis [10,20,50]. However, the inhibition e¡ect seems to depend on the bacterial strains and the concentration of nitrogen compounds applied. Generally, 6 100 WM nitrite, 1^2 WM NO and 6 1 mM N2 O were found to be su¤cient to completely inhibit H2 -de-

pendent methanogenesis [20,50]. Similar results were obtained for acetate-dependent methanogenesis [51]. Detailed experiments in Italian rice ¢eld soil have shown that it is mainly the toxicity of nitrite, NO and/or N2 O to methanogens that exerts the inhibitory e¡ect upon addition of nitrate to methanogenic rice ¢eld soil [10,20,21]. In addition, since nitrate addition decreases the H2 partial pressure to levels that methanogens are not able to utilize, competition for available substrates is believed to be an additional inhibitory mechanism [8,10]. In the present study, we demonstrated that nitrate addition completely inhibited methanogenesis from glucose and acetate (Figs. 2G and 4E). Since radioactive glucose or acetate were added when the reduction of nitrate was in progress (see Section 2), the denitri¢cation intermediates NO3 2 , NO and N2 O were present in the soil slurry at concentrations su¤cient to exert a toxic e¡ect on methanogens. Addition of nitrate decreased the glucose turnover rate constant (Table 1), drastically decreased the accumulation of radioactive acetate (Fig. 2C) and completely inhibited the production of 14 CH4 from [U-14 C]glucose (Fig. 2G). In the absence of nitrate, glucose in anoxic rice ¢eld soil is metabolized by fermentation [26,27]. The e¡ect of nitrate indicates that the glucose-fermenting microbial populations were inhibited by nitrate and/or its denitri¢cation products. For example, NO is highly reactive and therefore a non-speci¢cally acting toxic compound for microorganisms [52^54]. In addition, glucose may have served as electron donor for denitri¢er and thus have been completely oxidized to CO2 or assimilated into biomass [28,55]. In this case, however, the e¤ciency of glucose utilization by the nitrate-reducing microbial populations was less than that by the fermenting bacteria because of the relatively lower turnover rate constant. Nitrate addition resulted in stimulation of the acetate turnover constant (Table 2), even in the presence of chloroform, demonstrating that denitrifying bacteria consumed the acetate instead of methanogens when nitrate, nitrite, NO and/or N2 O became available. In the experiments with [2-14 C]acetate, addition of nitrate even resulted in a drastic decrease of the acetate concentration. Such a decrease was not observed in previous studies [10,21] and was also not observed in the experiments with [U-14 C]glucose. We have presently no explanation for this discrepancy. We have used in all the experiments the same batch of dry soil, only the slurry preparations (including breaking and sieving of the soil) were done at di¡erent times, though according to the same protocol. Addition of carrier-free [U-14 C]glucose or [2-14 C]acetate should not make a di¡erence. Therefore, we speculate that, due to the heterogeneous nature of soil, di¡erent microbial communities may have developed out of di¡erent soil or that we have a¡ected the soil during preparation in a way which we do not yet understand. Although the nitrate reducers utilized acetate more e¤-

FEMSEC 1089 27-12-99


ciently than the methanogens, we do not believe that the inhibition of 14 CH4 production from [2-14 C]acetate by nitrate was primarily caused by competition. Since the inhibition by nitrate was immediate, complete and irreversible, we conclude that toxicity was the main inhibitory mechanism for methanogenesis. If competition would have been the sole inhibitory mechanism, at least small amounts of 14 CH4 should have been detected, similar to observations in the presence of sulfate or ferrihydrite (see below). Previous studies have shown that addition of ferrihydrite and sulfate decreases the H2 partial pressure in anoxic rice ¢eld soil [8,9], as in aquatic sediments [56,57], and subsequently causes inhibition of methanogenesis. Unlike nitrate and its denitrifying products, ferric iron or sulfate ions are not toxic to methanogens. Although sul¢de, the product of sulfate reduction, may be toxic to methanogens, inhibition is only expected at elevated concentrations [58]. In Italian rice ¢eld soil, addition of 10 mM sul¢de did not inhibit CH4 production (unpublished results). Therefore, competition for available substrates was likely to be the exclusive mechanism for inhibition of methanogenesis by ferric iron and sulfate. Glucose may be utilized by ferric iron reducers, but Lovely [57] pointed out that the known species transfer only a small portion (1^3%) of the reducing power to ferric iron and mostly degrade glucose by fermentation. Our results for glucose consumption coupled with ferric iron and sulfate reduction also showed that there was no di¡erence in turnover rate constants and product formation patterns of glucose degradation in the presence and absence of ferric iron or sulfate (Table 1). Radioactive acetate accumulated to a similar level as in the control (Fig. 2C). Hence, it is likely that ferric iron reducers and sulfate reducers were not able to compete with fermentative microorganisms for glucose. Thus, only fermentation products of glucose metabolism such as acetate were utilized competitively with the methanogens. A previous study of Italian rice ¢eld soil showed that ferric iron or sulfate addition decreased the H2 partial pressure below the threshold concentration of methanogens [8,9], thus explaining the inhibition of H2 -dependent methanogenesis by competition. However, the ability of ferric iron and sulfate reducers to use acetate in rice ¢eld soil is still not clear. It was suggested that acetate-utilizing ferric iron reducers in rice ¢eld soil are able to utilize acetate but are not able to outcompete methanogens for acetate [9]. In one set of experiments (with [U-14 C]glucose), acetate concentrations indeed decreased only marginally upon the addition of ferric iron or sulfate (Fig. 3). This decrease would not be su¤cient to inhibit acetotrophic methanogenesis. In the other experiment (with [2-14 C]acetate), however, acetate concentrations decreased drastically (data not shown), indicating that acetate-utilizing ferric iron reducers did outcompete methanogens for acetate. Furthermore, acetate was oxidized mainly

83

to CO2 (RI = 0.86), demonstrating a diversion of the electron £ow away from CH4 production towards CO2 production. This diversion was also observed in the experiments with [U-14 C]glucose where much less 14 CH4 was formed although the maximum amount of radioactive acetate formed as intermediate was similar. Similarly, acetate-utilizing sulfate reducers have been found to be present only as spores and thus not active in acetate consumption [9]. Also, sulfate reduction was not stimulated by addition of acetate [9]. In the present experiments with [2-14 C]acetate, however, acetate turnover rate constants increased in the presence of sulfate and the acetate pore water concentration decreased, indicating that sulfate reducers consumed acetate more e¤ciently than the methanogens. Sulfate reducers also competed successfully for acetate in the experiment with [U-14 C]glucose, since less 14 CH4 was formed although the same amount of radioactive acetate accumulated transiently. In this case, however, acetate concentrations decreased only marginally. We assume that the populations of acetate-consuming anaerobes were di¡erent in the two experiments and, despite the fact that in both cases acetotrophic methanogenesis was partially replaced by acetotrophic iron or sulfate reduction, the underlying mechanisms were di¡erent. We found that acetate turnover was almost completely inhibited by the addition of chloroform both in the control and in the presence of sulfate (Table 2). In the control, inhibition of acetate consumption is due to the inhibition of methanogens by chloroform. In the presence of sulfate, inhibition of acetate consumption in rice ¢eld soil by chloroform has also been suggested to be due to the inhibition of methanogens by assuming syntrophic acetate oxidation coupled via interspecies H2 transfer between acetoclastic methanogens and H2 -utilizing sulfate reducers [9]. Indeed, H2 -dependent sulfate reduction has been found to be insensitive to chloroform inhibition [9,59]. However, recent investigation has shown that chloroform inhibits all bacteria using the acetyl-CoA pathway for acetate consumption, including acetate-utilizing Desulfotomaculum acetoxidans [59] and possibly also Desulforhabdus species. Therefore, the inhibition of sulfate-dependent acetate consumption by chloroform observed in the present study (Fig. 4B,D) can be attributed to acetate consumption by sulfate reducers using the acetyl-CoA pathway. This observation indicates that acetate-utilizing sulfate reducers were active in the present experiments. We can presently only speculate how bacteria reducing sulfate or ferrihydrite would outcompete methanogens without decreasing the acetate concentration substantially. Syntrophic acetate utilization is plausible in the case of sulfate reducers coupled to acetotrophic methanogens [9,23], but the same mechanism for a completely di¡erent physiotype such as iron reducers is unlikely. Conrad [14] recently pointed out that the relative population sizes of the competitors are important for causing a decrease in the acetate concentrations. If the populations of iron or sul-

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84


fate reducers would be small compared to those of the methanogens, addition of ferrihydrite or sulfate, respectively, would partially inhibit methanogenesis without causing a decrease of acetate. Hence, di¡erences in the populations and/or population sizes of iron and sulfate reducers between the experiments with either [U-14 C]glucose or [2-14 C]acetate may be the reason for the di¡erences in the acetate pools. Previous experiments have shown that the numbers of acetotrophic sulfate reducers may change quite dynamically for unknown reasons [24]. Competition between iron or sulfate reducers and methanogens for acetate also depends on the characteristics of the methanogenic populations. Italian rice ¢eld soil contains both Methanosarcina and Methanosaeta species as acetotrophic methanogens [60,61]. Recently, it has been found that the relative abundance of these two methanogenic genera changes dynamically with incubation time and incubation temperature [62]. Such a change will be important for the outcome of a competition for acetate, since the kinetic characteristics of the two methanogenic genera are completely di¡erent [63], i.e. the Km values and thresholds for acetate are much lower in Methanosaeta than in Methanosarcina species. Indeed, Methanosaeta was found to be as e¤cient an acetate utilizer as Desulforhabdus, while Methanosarcina was not [64]. In conclusion, our results demonstrate that glucose metabolism in rice ¢eld soil was not a¡ected by the presence of active ferric iron and sulfate reduction, but only by nitrate reduction which probably inhibited the glucose fermentation by producing toxic denitri¢cation intermediates. On the other hand, addition of all of these electron acceptors resulted in inhibition of methanogenesis from [2-14 C]acetate, most likely due to successful competition of methanogens for acetate. The fact that in some of the experiments, acetate was reduced to lower concentrations in the presence, than in the absence, of nitrate, iron or sulfate means that the bacteria reducing nitrate, iron and sulfate had a higher acetate-utilizing activity than the methanogens. Otherwise, for example at low population densities, the acetate concentration should not decrease [14]. Syntrophic acetate degradation, as concluded in previous studies [9,23], may also contribute to the observed patterns of acetate concentration and inhibition of methanogenesis by sulfate. Future studies have to address the dynamics of the microbial community structure in rice ¢eld soil to further elucidate the mechanisms of interaction between nitrate reducers, iron reducers, sulfate reducers and methanogens. Acknowledgements We thank Stefan Ratering for advice and help during the determinations of iron and the Fonds der Chemischen Industrie for ¢nancial support. Amnat Chidthaisong was

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